The present invention relates to bacteria that metabolize phenylacetate via a mandelate pathway. More particularly, the present invention relates to a new group of bacteria characterized by an ability to produce valuable compounds, including natural benzaldehyde, mandelate, benzyl alcohol, benzoate and cis,cis-muconic acid, which correspond to various intermediates along the aforesaid pathway.
A major use of benzaldehyde is as an ingredient in natural cherry flavors. An undesirable feature of the known processes for preparing benzaldehyde from products like apricot kernels or reground press cake, as disclosed in U.S. Pat. No 1,416,128, is that toxic hydrocyanic acid, along with benzaldehyde, is produced which must be separated completely from the benzaldehyde. U.S. Pat. Nos. 4,617,419, and 4,673,766, disclose the production of benzaldehyde from cinnamaldehyde according to a retro-aldol reaction, without production of toxic side products. But the disclosed process requires a source of cinnamaldehyde, which may have to be distilled or extracted prior to reaction.
A living organism that degrades a carbohydrate feedstock, or L-phenylalanine, along a catabolic pathway having a benzaldehyde intermediate could provide a source for the production of natural benzaldehyde. Earlier studies have suggested that pathways for the degradation of L-phenylalanine and phenylacetate along a mandelate pathway may occur in certain insects (Towers et al. (1972) Can. J. Zool. 50(7): 1047-1050), micro-algae (Landymore et al. (1978) Phycologia 17(3): 319-328), and fungi (Hockenhull et al. (1952) Biochem. J. 50: 605-609 and Bioprocessing Technology (1987) 9(11): 2-3).
However, it was generally understood heretofore that bacteria could not metabolize phenylacetate via a mandelate pathway, thereby to produce a desired intermediate like benzaldehyde. In this regard, the phrase "mandelate pathway" refers to that series of enzymatic degradations that converts mandelate to cis,cis-muconic acid. Intermediates of the degradation include benzoylformate, benzaldehyde, benzoate and catechol. In accordance with standard terminology, the names used to refer to acid intermediates reflect their actual form in vivo (i.e., in solution), thus mandelic acid is referred to as mandelate.
Such a pathway is known to exist in both Pseudomonas (e.g., Stevenson et al. (1964) Biochem. J. 96: 354-362) and Acinetobacter calcoaceticus NCIB 8250 (e.g., Cook et al. (1975) J. Gen. Microbiol. 91: 325-337), but it is only revealed when the bacteria are provided with feedstock in the form of mandelate or some other intermediate further along the pathway. Mandelate or benzoylformate are not readily available as natural products. These and other intermediates are also too expensive to be of use as feedstocks in a commercial process for subsequent intermediates.
One early study tested the ability of a wide range of aromatic compounds, both metabolizable and nonmetabolizable, to induce enzymes of the mandelate pathway. (Hegeman (1966) J. Bacteriol. 91(3): 1140-1154). Phenylacetate and phenylpyruvate, both possible degradation intermediates of L-phenylalanine, were among the many compounds tested and were shown to induce activities for mandelate dehydrogenase and benzoylformate decarboxylase. But the link which allowed conversion of phenylacetate to mandelate was completely unknown, and available biochemical evidence shows that L-phenylalanine and phenylacetate are not metabolized through catechol to ketoadipate (Wheelis and Stanier (1970) Genetics 66: 245-266).
Consequently, the conversion of phenylacetate to mandelate by bacterial action has been considered infeasible heretofore, even though expression of such a pathway in bacteria could, in principle, enable the accumulation of valuable chemical compounds as intermediates, for example, of microbially mediated L-phenylalanine degradation.